Quantitative equilibrium binding studies of the nonenzymatic protein-protein interactions underlying the role of factor Va in the mechanism and regulation of human prothrombin activation are proposed. The interdependent interactions among factor Xa (enzyme), prothrombin (substrate), factor Va (cofactor) and the surfaces of blood platelets are required to achieve the rate of prothrombin activation necessary to support normal hemostasis. The importance of these cofactor-mediated interaction in the regulation of normal blood coagulation is underscored by the correlations between decreased levels of anticoagulant blood enzymes which act on factor Va and reactions of enzyme inhibitors which are modulated by factor Va, and an increased tendency towards thrombosis. The proposed investigation is directed at a quantitative description of the enzyme-cofactor and protein substrate-cofactor binding interactions which represent the unique primary biochemical functions of factor Va and which, in the absence of surfaces, are responsible for the assembly of the multicomponent complexes producing thrombin at the greatest rate. To quantitate these interactions, an approach based on fluorescence measurement of proteins labeled with fluorescent dyes and used as binding probes is proposed. Versatile new methods of active site fluorescence labeling of proteases will be used to obtain probes which provide fluorescence spectral changes by which to monitor the formation of complexes with factor Va. Development of these probes will enable binding between the native proteins to be quantitated as well as the binding of related species which compete for the same sites on the cofactor, such as the prothrombin activation intermediates. Quantitation of the competition and cooperation between the cofactor interactions is essential to interpreting the kinetics of the factor Va-accelerated reactions of prothrombin activation. The role of cofactor binding in the mechanism of the acceleration of two of the reactions leading to thrombin formation and how the cofactor may control the activation pathway by determining the specificity of factor Xa for alternate substrates will be investigated by comparison of the kin-etic behavior of the reactions with the formation of complexes established from binding measurements.